The protein expression facility (PEF) functions as a support group for the principle investigators at the NIEHS. Our focus is to provide the research groups at the NIEHS with a means to generate the proteins they require so that they can perform their experiments. These projects range from generating enough of a given protein to perform structure function studies, to the generation of protein fragments for anti-body generation, to creating stable cell lines for more in vivo assays. Each new protein that is expressed in E. coli is tested using four different n-terminal tags (6 x His, 6 x His-Thioredoxin, 6 x His-glutathione S-transferase, and 6 x His-Maltose binding protein). Initial expression testing is done in Rosetta2(DE3) pLacI cells at 18C and 30C. These tags help to fold/solubilize the protein of interest and provide a uniform initial purification step that helps to identify which combination of tag and temperature yield the most of the desired product. The Rosetta cell line helps remove any codon bias against expression in E. coli. Once expression is demonstrated and a tag selected other variables such as alternative cells lines, media and temperature can all be optimized for the best yield. If expression in E. coli fails to work or is not feasible due to the need for post translational modifications, then baculovirus/insect cell expression system is tried. Expression is investigated using three n-terminal tags (6 x His, 6 x His-glutathione S-transferase, and 6 x His-Maltose binding protein) using two cell lines (SF9 and High Five). Expression trials are carried out at both 27C and 20C. All new baculovirus that are generated are tittered using the Sf9et cells to demonstrate infectivity as well as determining viral titer. Once the system is established, all future scale up is done using TIPs protocol to save time, storage space, and provide for long term storage of the baculovirus at -135C. The PEF also has vectors available for expression of protein in mammalian cell lines. Expression is tested in Cos-7, HEK293, CHO and/or Hela cells unless a more unique cell line is desired by the principle investigator. Expression is first tested transiently followed by the generation of a stable cell line, if this is possible/desired. If the above methods yield a protein that is only in insoluble aggregates (inclusion bodies), then protein refolding can be attempted. Each refolding project is tested using both rapid dilution and a high hydrostatic pressure approach. The rapid dilution approach is performed in a 96 well multi matrix format while the high hydrostatic approach uses a more sequential multi-sample (20 sample per run) approach. Project examples: Allergenic protein initiative: Robert London and Geoff Mueller The NMR group has begun a series of structure function studies on a group of proteins which are known allergens. To support this effort, the PECF has had the genes of target proteins (Ara h2, Bla g2, Bla g6, Der p 2, Der p5, Der p 7, Scfv, Can f 5, Cat R 1, Rage) synthesized (this saves time, money, and effort on our part) for expression in E. coli and baculovirus. Expression in E. coli proved sufficient in all cases to obtain enough protein for x-ray crystallography and NMR structure studies; however, lipopolysaccharides derived from the bacteria complicated functional analysis in the allergen assays. In order to pursue this line of study, the proteins were express and purified from insect cells using baculoviruses. Fms Related Tyrosine Kinase 3 ligand (FTL3-l): Don Cook and Jennifer Martinez FTL3-l was requested by two different groups in the IIDL (Immunity, Inflammation, and Disease Laboratory). This protein is generally used in cell based assay to look at innate immunity response in dendritic cells. Both groups needed the protein to use as a reagent in their studies. The PECF had the gene synthesized for expression in E. coli. We then worked out a refolding protocol that yielded mgs of active protein that was endotoxin free for use in their cell assays. This amount of protein allows both groups to proceed with their studies over lengthy periods of time without batch to batch variations as they had seen in commercial sources. Antibody interaction with Ara h2: Robert London, and Lars Pederson Ara h2 is one of the proteins involved in peanut allergies. Investigators at the NIEHS are collaborating with allergy physicians to look at why some patients respond to new immune therapies to Ara h2 allergies and some do not. To examine this several antibodies to Ara h2 were isolated from patients and then 5 were selected based on response groupings to the therapy. These antibodies were sent to the NIEHS investigators for structure function studies (how do the antibodies differ in their interaction with Ara h2). The PEF already had the Ara h2 protein expressed and purified (see above). Although the PEF had previously demonstrated a PEI/HEK293 system would produce the desired anti-body, several expression constructs failed to make enough anti-bodies for the desired study. The PEF has switched to the expiCHO-s expression system, which yielded greater than 350 mg/L of purified anti-body in most cases. These improved yields enabled the researchers to proceed with thier studies. Mammalian expression of proteins for the Stanley Group The Stanley Lab frequently utilizes the support of the Protein Expression Core/Structural Biology Core for assistance with mammalian cell culture including large scale protein expression and small scale co-immunoprecipitations. For example, to characterize the association between the essential AAA-ATPase NVL2 and the ribosome assembly factor WDR74 the Core performed a series of transient transfections of different truncations and variants of these two proteins. Co-immunoprecipitations with the cores in-house made GFP resin were then used to assay for protein association. Using this methodology, the Stanley lab was able to determine a unique method of association of the AAA-ATPase motor protein and its substrate. Expression of large protein complexes for the Wade Group The PEF is determining the optimal method for expression of the NuRD complex. Each of the target proteins have been expressed using single target baculoviruses. Mixing the single target baculovirus failed to yield enough of the complex as desired by the Wade group. The Multibac system failed to yield the desired large complexes, but the Macrobac system worked well. We currently have the capability of expressing any combination of the NuRD complex proteins, including the entire 5 protein complex using a single baculovirus particle. Studies are underway to determine where purification tags can be used to purify the desired complexes up to and including the entire 5 protein complex. Expression of protein standards for Cryo-EM studies and the Borgnia Group: The PEF has produced both the 20S proteasome from Thermophilus acidophilum and apo-Ferritin as test molecules for the new Cryo-EM facility at the NIEHS. Both proteins are currently being used to help people learn how to prepare grids, collect data, and solve structures using Cryo-em. The apo-Ferritin structure has been refined to 3.1 angstrom resolution on the Arctica instrument. The PEF is working on using the apo-Ferritin as scaffold molecule for to display small proteins for Cryo-em structure determination. This project was successful on test molecules and we are working with the Borgnia group on improving the tether length, data collection and structure determination methods. The PEF has worked on 86 different projects for 25 different PI groups from 6 different Laboratories within DIR as well as research groups from the CRU and the DNTP.